The ability to develop and sustain memory CD8 T cells after infection or immunization is a hallmark of the adaptive immune response and one basis for protective vaccination against infectious disease or in cancer immunotherapy. Since the degree of protection to infection depends on the functional characteristics (quality) and the number (quantity) of memory CD8 T cells present at the time of pathogen exposure, understanding the mechanisms that govern generation, differentiation and maintenance of the memory CD8 T cell pool are critical to our ability to design the most effective vaccines. Substantial progress has been made in our understanding of the biology of memory CD8 T cells generated after acute infection or immunization. However, despite this progress many important questions remain. For instance, the superior protective capacity of memory CD8 T cells is closely linked to their increased abundance in both lymphoid and non-lymphoid organs, and as a consequence much effort has been devoted to identifying strategies that increase the absolute numbers of memory CD8 T cells. Among these strategies, prime-boost regimes (or multiple antigen (Ag) stimulations) are often used because of their ability to elicit large numbers of memory CD8 T cells. Importantly, our recent studies demonstrated that each additional re-stimulation with Ag results not only in progressive decrease in proliferative capacity of the ensuing memory CD8 T cell populations but also change their phenotype, function, rate of contraction, basal proliferation and ability to survive. Interestingly, these functional changes ar associated with transcriptomic diversification in memory CD8 T cells after each antigen encounter. However, it is unknown which key molecular mediators are responsible for each of these functional changes. Filling this knowledge gap has critical importance in order to optimize memory CD8 T cell numbers while preserving qualities that impact protection. Our long-term goal is to fully understand the functional consequences imposed on memory CD8 T cell populations generated after one or more Ag encounters. This information will be significant in formulating the best strategies to generate and manipulate protective CD8 T cell-mediated immunity in response to vaccination. We will test the overall hypothesis that the history of Ag-stimulations is a critical determining factor controlling the function and long-term maintenance of memory CD8 T cell populations. We will begin to address our long- term goal through the following Specific Aim - Determine the molecular mechanisms controlling differentiation and function of memory CD8 T cells generated after repetitive antigen encounters.